Cancer-associated fibroblasts as facilitators in nasopharyngeal carcinoma progression via exosomal MMP13

Abstract

Background

Cancer-associated fibroblasts (CAFs), essential components of the tumor microenvironment (TME), contribute to tumor formation. Our previous research demonstrated that exosomes containing matrix metalloproteinase 13 (MMP13) accelerate metastasis in nasopharyngeal carcinoma (NPC). This study aimed to determine whether exosomes containing MMP13 promote NPC development by stimulating the differentiation of normal fibroblasts (NFs) into CAFs.

Methods

The presence of CAFs in NPC was identified through immunohistochemistry (IHC) analysis. NPC cells activated NFs into CAFs, according to the co-culture cell model. Exosomes were characterized by Western blot and electron microscopy analysis. NFs were treated with exosomes, and Western blot, migration, and 5-ethynyl-2’-deoxyuridine (EdU) assays were conducted to assess CAFs activation. Western blot analysis was used to assess whether exosomes derived from CAFs activated the Notch signaling pathway in human low-differentiated nasopharyngeal carcinoma cell line (CNE2). The effect of MMP13 on exosomes generated from CAFs in vivo was verified using a nude mouse model, and the effect of MMP13 on the immune microenvironment of CAFs in vivo was verified using the C57BL/6 mouse model.

Results

We found that NPC cells, after converting NFs to CAFs, exhibited enhanced proliferative and migratory capacities. NPC cells secreted MMP13 containing exosomes, which facilitated the conversion. The secretion of exosomes by activated CAFs has been demonstrated to promote tumor progression by activating the Notch signaling system, promoting angiogenesis, and suppressing T cell immunity. The inhibition of exosome MMP13 has the potential to impede the migration and proliferation of recipient cells. Inhibition of exosomal MMP13 may suppress recipient cell migration and proliferation.

Conclusions

Our research identified a critical mechanism in which MMP13-rich exosomes from NPC cells may influence the development of CAFs in the TME, while the exosomes produced by CAFs further support the malignant development of NPC.

Highlight box.

Key findings

• The exosomal matrix metalloproteinase 13 (MMP13) secreted by nasopharyngeal carcinoma (NPC) cells contributes to the conversion of normal fibroblasts (NFs) to cancer-associated fibroblasts (CAFs), and the activated CAFs accelerate tumor progression by activating the Notch signaling system.

What is known and what is new?

• NPC metastases were accelerated by exosomal MMP13.

• MMP13-rich exosomes accelerated NPC development by stimulating the differentiation of NFs into CAFs.

What is the implication, and what should change now?

• This study defined a crosstalk mechanism between NPC and NFs cells that stimulates tumor growth and suppresses T-cell immunity via the MMP13-rich exosomes, which may support effective NPC prevention and therapy approaches.

Introduction

Nasopharyngeal carcinoma (NPC), an epithelial squamous cell cancer, is prevalent in southern China and is associated with poor prognosis and high relapse rates (1). The prognosis for NPC patients is still bleak despite significant attempts to develop treatments. Therefore, more investigation into the molecular mechanisms driving NPC metastasis is crucial.

Exosomes, which play a significant role in the occurrence, development, recurrence, and metastasis of malignant tumors, are one of the mechanisms through which tumor cells interact with stromal cells (2). Exosomes are membrane-enclosed vesicles approximately 100 nanometers in diameter, produced by nearly all cell types (3,4). Exosomes transfer biological information including microRNAs (miRNAs), proteins, lncRNAs, metabolites, and other chemicals, which has been shown to have biological and therapeutic effects in numerous study (5). In previous study, we found that matrix metalloproteinase 13 (MMP13), rather than MMP2 or MMP9 was overexpressed in NPC-derived exosomes and regulated the metastatic and angiogenic capacity of the human low-differentiated nasopharyngeal carcinoma cell line (CNE2) (6). Exosomes also facilitate communication between tumor and stromal cells by transmitting MMP13, such as between vascular endothelial cells and fibroblasts (6). However, the mechanism remains poorly understood. This study investigates the critical role of fibroblast-NPC cell interactions.

The tumor microenvironment (TME) plays a constructive role in cancer progression (7,8). Activated cancer-associated fibroblasts (CAFs), as core components of TME, are remarkably heterogeneous, with different subtypes exerting different regulatory functions in immune response, angiogenesis, metabolism, and extracellular matrix (ECM) remodeling (9). These subtypes include tumor-promoting subtypes, such as myofibroblast-type CAFs (myCAFs) and inflammatory CAFs (iCAFs), and tumor-suppressor subtypes, such as interferon-responsive CAFs (ifCAFs) (10). At present, there is a paucity of specific markers that can reliably distinguish between the various subtypes of CAFs, its function has not been subdivided in NPC. This study utilized a series of specific markers for CAFs, including α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and fibroblast secretory protein 1 (FSP-1) (11). It has been hypothesized that CAFs interact with cancer cells to facilitate invasion, metastasis, and progression (12). However, the mechanisms by which tumor cells activate CAFs in NPC are still mostly unclear.

MMP13, a member of the matrix metalloproteinase family, is critical for remodeling ECM and facilitating the emergence, growth, invasion, and metastasis of malignant tumors (13). MMP13 is highly expressed in a variety of tumors, ranging from melanoma (14), breast cancer (15), and ovarian cancer (16). Numerous studies have found that exosomes secreted by CAFs contain a variety of molecules that promote tumor progression compared to normal fibroblasts (NFs). Qu et al. found that CAFs-derived exosomal DACT3-AS1 is a suppressive regulator in malignant transformation and oxaliplatin resistance (17). Lu et al. found that CAFs-derived exosomal TUG1 promotes migration, invasion, and glycolysis of HCC cells through the miR-524-5p/SIX1 axis (18). However, MMP13’s function in exosomes derived from NPC cells and CAFs has not been fully elucidated.

This study focuses on the role of MMP13 in exosomes and explores the interaction between NPC cells and CAFs. On the activation of CAFs and the advancement of NPC, respectively, we will focus on the impacts of exosomal MMP13 generated from NPC cells and CAFs. When taken as a whole, this research may provide new insights into NPC molecular targeted therapy and prognosis assessment. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-428/rc).

Methods

Cell culture

The normal nasopharynx epithelial cell line NP69 and the human NPC cell line CNE2 was a gift from Sun Yat-sen University and Xiangya School of Medicine. The human breast cancer cell line MDA-MB-231, the lung cancer cell line H1299 and murine LLC cells were purchased from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). RPMI-1640 medium with exosome-depleted FBS (Gibco BRL, XXXX, USA) was used for culturing CNE2 cells, MDA-MB-231 cells, H1299 cells and LLC cells. In a predetermined keratinocyte serum-free medium, NP69 cells were grown (Invitrogen, Carlsbad, CA, USA). As previously described (19), NFs were derived from normal nasopharynx tissues. In short, fresh tissues were sliced into pieces of 2–3 mm pieces and cultivated in a high-glucose Dulbecco’s Modified Eagle Medium (DMEM) medium (Gibco) until fibroblasts appeared. Cells were cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum (FBS, ScienCell, XXX, CA, USA),100 U/mL penicillin, and 100 U/mL streptomycin (Thermo, HYCLONE, XXX, USA). All cells were cultured in a humidified incubator containing 5% CO₂ at 37 ℃.

Human tissue samples

This study was performed in line with the principles of the Declaration of Helsinki and its subsequent amendments. With the approval of the Ethics Committee (IRB No. 2017-L078), tissue samples were collected from 8 patients with pathologically confirmed NPC and 8 normal donors at the Affiliated Hospital of Nantong University. Each patient was fully informed and signed a consent form before performing the experiments.

Exosomes isolation and labeling

Under various circumstances, we extracted the conditioned media from cultivated cells (20). We collected the supernatant after centrifuging the medium at 500, 3,000, and 10,000 g sequentially. The exosome pellet was created by ultracentrifuging these supernatants at 100,000 g for 90 min. Following a wash, the pellets were resuspended in 20 µL of phosphate-buffered saline (PBS). Suspended exosomes (50 µg) in 1 mL of PBS to determine how cells absorbed the exosomes. Receptor cells were fixed after the exosomes were co-cultured with PKH67 membrane dye (Sigma-Aldrich, XXX, XXX) for 30 min. After that, we used Hoechst to highlight the nucleus in the dyed cells. A confocal microscope is used to take the photographs 10 min later.

Nanoparticle tracking analysis

Real-time vesicle characterisation was performed using a Zetaviewer nanoparticle tracking analysis system (Particle Metrix, XXX, Germany). Samples were injected into a pre-calibrated Zetaviewer, the 488 nm laser was selected, and ZetaView-315 was recorded. Filtered PBS was used as a blank control. The overall results represent the average vesicle size.

Electron microscopy

The 2.5% glutaraldehyde was used to fix the isolated exosome pellet. Cut the sample into 0.12-µm sections and stain with 0.2% lead citrate. On formvar grids made of copper mesh, the pellets were fastened. We examine samples with the JEOL transmission electron microscope (JEM-1230; JEOL, Tokyo, Japan).

Lentivirus production and infection

GeneChem (Shanghai, China) created lentiviral particles that carried the LV-MMP13-shRNA vector and their flanking control sequence (Mock for short). Lentiviral vector was used to infect CNE2 cells, and a Western blot verified MMP13 expression.

Immunohistochemistry (IHC)

All NPC samples and tissues from the healthy nasopharyngeal epithelium were obtained from the Nantong University Affiliated Hospital. We soak the tumor tissue in a fixative solution. Subsequently, these tissues were made into 4um paraffin sections. We used IHC to analyze the expression of Ki67, α-SMA and CD34 in tissues. The samples were then marked with 3,3’-diaminobenzidine (DAB) chromogen. Positive cell proportion was categorized into four classes based on staining intensity: 1 (negative), 2 (weakly positive), 3 (moderately positive), and 4 (strongly positive). For staining intensity, the grades were 1 (0–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). The staining score was calculated by adding the two scores; 0–8 indicated low expression of α-SMA, and 9–16 indicated strong expression.

Immunofluorescence

On the coverslips of a 24-wells plate, we initially planted the cells that would be put to the test. We removed the cell slide and submerged it for 30 min in 4% paraformaldehyde. We applied the primary antibody (α-SMA, Abcam, XXX, USA, 1:50) to the cell slide after blocking it in the blocking solution, leave it overnight at 4 ℃, and then treated with the secondary antibody after PBS washed the slides. Nuclei were visualized by Hoechst. Finally, used a fluorescence microscope to detect and evaluate antibody expression.

Western blot analysis

To obtain the protein supernatant, we centrifuged the protein at 13,000 g for 15 min. The protein concentration is then measured and examined using the bicinchoninic acid (BCA) method. The protein was separated on the electrophoresis gel and moved to a 0.22 µm polyvinylidene difluoride (PVDF) membrane. After that, membranes were treated overnight by primary antibodies from Abcam (USA) against α-SMA, FSP-1, CD9, TSG101, flotillin1, MMP13, Notch1, Notch3 and GAPDH. The secondary antibodies are then incubated after washing the target bands. The time of tablet pressing was calculated using the protein bands’ fluorescence intensity following the addition of electrochemiluminescence (ECL) luminous liquid.

Quantitative polymerase chain reaction (PCR)

We initially extracted total RNA from cells using Trizol (Invitrogen). After that, we created cDNA using a reverse transcription tool from Thermo Fisher. Then, we used SYBR Green Master Mix (ABI, XXX, USA) to quantify gene expression by PCR (qPCR). All primers are designed by the Ruibo reagent company. To determine the expression level, the target gene’s Ct value is compared to that of the reference gene (GAPDH).

Transwell migration

We placed the 8.0 µm pore size chambers in a 24-well plate. We added 10 percent FBS to the culture medium in the lower chamber as we injected the digested 5×10⁴ cells into the top chambers using the medium without FBS. We took the chambers out for fixing and coloring after around 20 hours. We cleaned the chamber’s bottom and wiped the inside with cotton swabs. The cells on the bottom of the basement membrane were fixed, dyed in 0.20% crystal violet. Five cells per well were randomly counted under light microscope. In terms of cell counts, the average count of cells in each field of view was used.

Cell viability assay

The logarithmic growth phase cells were planted onto 96-wells plates, and the Cell Counting Kit-8 (CCK-8) reagent was applied at 8-time intervals every 12 hours. We checked the absorbance of each well on the 96-well plate while debugging the microplate reader to 450 nm.

5-Ethynyl-2’-deoxyuridine (EdU) cell proliferation detection

We seeded 96-well plates with cells in good growth state at a density of 1×10⁴ per well. After EdU labeling, cell immobilization, Apollo staining, and DNA staining, we observed the cells.

Animal xenograft tumor model

Four-week-old male BALB/c nude mice and C57BL/6 mice were provided by the Laboratory Animal Center of Nantong University. Tumor cells (CNE2 and LLC cells 3.0×10⁶ cells) were implanted subcutaneously in the ventral side of nude mice. Tumor volumes (mm³) were measured and estimated at five-day intervals using the following equation: volume = 0.5 × width² × length. When the tumor reached a volume of 60 mm³ (approximately 8 days after tumor inoculation), the mice were randomly assigned to two groups, each consisting of 5 mice. The groups were injected subcutaneously with 10 µg of exosomes: 10 µg of CAFs exosomes, and 10 µg of shMMP13 CAFs exosomes that were absorbed in the tumor xenografts. The mice were put to death after receiving 5 injections, and the tumors were analyzed. A protocol was prepared before the study without registration. Animal experiments were performed under a project license (No. 20190304-005) granted by the Institutional Animal Care and Research Advisory Committee of Nantong University and in compliance with institutional guidelines for the care and use of animals.

Ultrasound Doppler

Following the administration of isoflurane gas to induce anesthesia in the nude mice, the perfusion of the subcutaneous tumors was estimated by power Doppler imaging, and the ratio of microvessels within the tumors was analyzed using the HERA W10 color Doppler diagnostic ultrasound machine (Samsung, Korea) equipped with a CA2-9A probe operating at a frequency of 50 MHz.

Flow cytometry

Lymph nodes were harvested from C57BL/6 mice and processed through a cell strainer (40 µM pore size) and resuspended in PBS. For quantification of dendritic cells, lymph nodes were first chopped into small pieces and incubated in collagenase IV (1 mg/mL, C5138, Sigma) and DNase I (0.2 mg/mL, Roche, XXX, XXX) for 30 min at 37 ℃ with gentle agitation. Following digestion, cells were passed through a 40 µm strainer and resuspended in PBS supplemented with 2% FBS (Gibco). A 20 min blocking step using anti-CD3, CD19, CD4, CD8 antibodies (BD Biosciences, XXX, XXX) were performed on ice prior to fluorescent staining and analyzed by flow cytometry using a FACSCanto II flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (version XXXXXX).

Statistical analyses

Data were analyzed using Student’s t-test, one-way, and two-way analysis of variance (ANOVA) after each experiment was carried out at least three times, and the results were expressed as the mean ± standard deviation (SD). The X-tile software (version XXXXXX) examined the staining score. The differences between groups were deemed statistically significant when P<0.05.

Results

Co-cultured with NPC cells activates NFs into CAFs

CAFs have been shown to contribute to tumor development. α-SMA, the most effective marker, is overexpressed in various cancers. Yu et al. demonstrated that α-SMA was overexpressed in NPC (21). Additionally, high α-SMA expression in CAFs predicts poor prognosis and had an impact on overall survival (OS), progression-free survival (PFS) primarily and distant metastasis-free survival (DMFS) (21). Elevated α-SMA expression was observed in the tissues of NPC patients (Figure 1A,1B). A co-culture cell model was created to further investigate CAF activation (Figure 1C). The low differentiation NPC cell line CNE2 cells were positioned in the upper chamber of a six-well plate, with NFs in the lower chamber. After co-culturing for 1 week, cell morphology was observed under a light microscope. The cells in the lower chamber were spindle-shaped, with increased antennae, which were considered as the typical morphology of CAFs (Figure 1D). Western blot and reverse transcription-PCR (RT-PCR) were used to further identify the CAFs markers, revealing that α-SMA and FSP-1 were elevated at the protein and RNA levels, respectively (Figure 1E,1F). Biological differences between NFs and CAFs were further investigated using transwell and EdU experiments. As seen in Figure 1G-1J, CAFs exhibited enhanced migratory and proliferative capacities compared to NFs. Collectively, these results suggested that CAFs play a potential role in NPC tumor promotion.

Figure 1.

Co-cultured with NPC cells activates the activation of NFs into CAFs. (A) Typical images of staining in clinicopathologic sample of α-SMA from normal and nasopharyngeal carcinoma patients. (B) Quantification of IHC staining for α-SMA expression. (C) Scheme representing the model of NFs (lower) co-cultured with CNE2 cells (upper). (D) Representative images of cell shapes were observed by a light microscope after fixing NFs and CAFs with 4% paraformaldehyde. The arrows indicated the spindle-shaped, and the antennae increased shape of cells. The protein (E) and mRNA (F) expression of α-SMA and FSP-1 in NFs and CAFs. (G,H) Use the transwell assays to detect the migration ability of NFs and CAFs and quantitatively evaluate the number of migrating cells by crystal violet staining. (I,J) Representative micrographs of EdU-incorporating different cells (NFs and CAFs). **, P<0.01; ***, P<0.001. α-SMA, α-smooth muscle actin; CAF, cancer-associated fibroblast; CNE2, human low-differentiated nasopharyngeal carcinoma cell line; EdU, 5-ethynyl-2’-deoxyuridine; FSP-1, fibroblast secreted protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHC, immunohistochemistry; NF, normal fibroblast; NPC, nasopharyngeal carcinoma.

NPC cells exosomes mediate the activation of NFs into CAFs

Exosomes are considered a novel means of facilitating cell-to-cell communication. However, whether exosomes from NPC cells are involved in the activation with NFs to CAFs is still largely unknown. Exosomes were isolated from the conditioned media of NP69 and CNE2 cells. The double-layer closed shape of the exosomes was observed using transmission electron microscopy and dynamic light scattering with their size being measured (Figure 2A,2B). Fluorescence microscopy showed that PKH67-labeled exosomes isolated from the conditioned medium (CM) of CNE2 cells could be efficiently internalized by NFs (Figure 2C). In addition, Western blot analysis of CNE2-derived exosomes showed specific exosomes markers, including CD9 and TSG101, at the protein level (Figure 2D). Our immunofluorescence results support the depiction of α-SMA expression in Figure 2E. When compared to exosomes secreted by NP69 cell, adding exosomes derived from CNE2 cells for 3 days could upregulate the expression of α-SMA of NFs treated with CNE2 exosomes. Our findings suggested that exosomes derived from CNE2 cells may promote the activation of NFs into CAFs. No discernible changes were observed in the indicators after 12 hours of treatment with various types of cell exosomes, according to Western blot data (Figure 2F). However, after 3 days of culture using CNE2-derived exosomes, the expression of α-SMA was markedly increased (Figure 2G). Additionally, NFs that were activated into CAFs after being cultured with exosomes derived from CNE2 exhibited greater migratory abilities than those that were treated with exosomes produced by NP69 (Figure 2H,2I). These results suggest that exosomes from CNE2 cells exosomes mediate the activation of NFs into CAFs.

Figure 2.

NPC cells exosomes mediate the activation of NFs into CAFs. (A) Representative electron microscope image of exosomes released by CNE2 cells. Arrow indicated that the representative cup-shaped structure. (B) NanoSight analysis showed a mean particle size of 50–120 nm diameter structures that are typical of exosomes. The vertical line meant the relative concentration and the horizontal line meant the size (nm). (C) Typical microscope images represented the internalization of PKH67-labeled tumor exosomes in NFs. (D) The presence of exosomal markers was detected by Western blot. (E) Fluorescence representative images of α-SMA (green) in NFs cultured for three days with PBS, NP69, and CNE2-derived exosomes. (F,G) Detection of α-SMA protein expression levels by Western blot. (H,I) Analysis of cells migration by transwell assays. **, P<0.01. α-SMA, α-smooth muscle actin; CAF, cancer-associated fibroblast; CM, conditioned medium; CNE2, human low-differentiated nasopharyngeal carcinoma cell line; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NF, normal fibroblast; NPC, nasopharyngeal carcinoma; PBS, phosphate-buffered saline.

MMP13 containing exosomes regulates the biological behavior of CAFs in vitro

Exosomes have the capability to transport proteins and miRNAs to adjacent cells. Consequently, we next investigated the underlying mechanism by which exosomes originating from NPCs activate NFs into CAFs. Our previous research demonstrated that exosomes isolated from the peripheral plasma of NPC patients contained MMP13 and promoted NPC metastasis (6). Then, we utilized lentivirus knockdown MMP13 in CNE2 cells, as the protein level in these cells was higher than in NP69 cells (Figure 3A). We identified the expression level of MMP13 protein in exosomes from various treatment groups. Figure 3B demonstrates that the expression of MMP13 in exosomes from CNE2 was greater than that in exosomes from NP69. As a result of knocking down the cellular level of MMP13, the expression of MMP13 in exosomal protein also decreased. We also discovered that the markers of CAFs (α-SMA and FSP-1) were lower in NFs co-cultured with exosomes containing lower levels of MMP13 (Figure 3C). This might suggest a possible connection between MMP13 and CAFs activation. Transwell assay results indicated that migration rates correlated with MMP13 expression levels (Figure 3D,3E). EdU proliferation assays confirmed that NFs treated with low MMP13-low exosomes exhibited comparable outcomes to those treated with NP69-derived exosomes (Figure 3D,3F). Immunofluorescence analysis further supported the role of exosomal MMP13 in CAFs activation (Figure 3D).

Figure 3.

MMP13 in CNE2-derived exosomes regulates the biological behavior of CAFs in vitro. (A) Western blot analysis of MMP13 protein levels in NP69 and CNE2 cells. MMP13 knockdown is also verified. (B) The protein expression level of MMP13 represented the exosomes. (C) After co-culturing with exosomes from different cells, the protein expression level of MMP13 was detected by Western blot. (D) After treatment with exosomes derived from different groups, the migration and proliferation ability of NFs was evaluated by transwell and EdU assays. Representative immunofluorescence microscopy images show the typical markers (α-SMA) (green) of CAFs. Quantitative assessment of the number of migrating cells (E) and the ratio of EdU-positive cells (F). *, P<0.05; **, P<0.01. α-SMA, α-smooth muscle actin; CAF, cancer-associated fibroblast; CNE2, human low-differentiated nasopharyngeal carcinoma cell line; EdU, 5-ethynyl-2’-deoxyuridine; FSP-1, fibroblast secreted protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NF, normal fibroblast.

MMP13 in CAFs-derived exosomes facilitates the progression of NPC in vitro via the Notch signal pathway

CAFs-derived exosomes (generated from NFs activated by CNE2 exosomes) were collected and co-cultured with CNE2 cells. Co-uptake experiments revealed that CNE2 cells effectively internalized CAFs-derived exosomes (Figure 4A). We aimed to do additional research to ascertain MMP13’s impact on CAFs-derived exosomes. We used the CCK-8 and transwell assays to determine whether CAFs-derived exosomes could enhance NPC cells to proliferate and migrate. CNE2 cells treated with CAFs-derived exosomes showed increased proliferation and migration compared to cultures with NFs-derived exosomes (Figure 4B-4E). Similar phenomena were observed in the MDA-MB-231 and H1299 cells by transwell and EdU assays (Figure S1A-S1F). Lentiviral-mediated knockdown of MMP13 in CAFs resulted in exosomes with reduced MMP13 levels. Culturing cells with exosomes from shMMP13 CAFs inhibited cell proliferation and migration (Figure 4C,4D). Notch signaling has been demonstrated to play a pivotal role in regulating various biological processes, including cell proliferation (22), invasion, matrix remodeling, and immune landscape alterations. In light of these findings, we postulated that MMP13 might function as a critical mediator of CAF activation through this specific pathway. In contrast to NFs-derived exosomes and PBS, results indicated higher levels of Notch1 and Notch3 proteins in CNE2 cells cultured with exosomes derived from CAFs (Figure 4F), indicating the participation of the Notch signal pathway. Elevated levels of Notch1 and Notch3 proteins were also found in MDA-MB-231 cells cultured with exosome-incubated CAFs, but Notch3 was less pronounced in H1299 cells (Figure S1G). Research indicates that the secretion of MMP13 by CAFs promotes malignant phenotypes in various cancers. However, it is unclear whether the Notch pathway is activated in all cancer cells, particularly in NPC and breast cancer.

Figure 4.

MMP13 in CAFs-derived exosomes facilitate the progression of NPC in vitro via the Notch signal pathway. (A) Representative image of PKH67-labeled exosomes (green) derived from CAFs internalized by CNE2 cells. (B,C) Cell viability assays of CNE2 cells co-cultured with exosomes derived from CAFs with different MMP13 expression levels. (D) The migration ability of CNE2 cells was quantified after treatment with exosomes derived from CAFs with different MMP13 expression levels by crystal violet staining. (E) Quantitative assessment of the number of migrating CNE2 cells. (F) The protein expression of MMP13, Notch1 and Notch3 in the co-culture model under different conditions. *, P<0.05; ***, P<0.001; ****, P<0.0001. CAF, cancer-associated fibroblast; CNE2, human low-differentiated nasopharyngeal carcinoma cell line; NC, XXXXXXX; OD, optical density.

MMP13 in CAFs-derived exosomes promotes angiogenesis and progression of NPC in vivo

Exosomal MMP13 from CAFs was utilized in an in vivo xenograft model in nude mice to further investigate the impact of the exosomal MMP13. Ultrasound Doppler showed that injection of shMMP13 CAFs-derived exosomes reduced the proportion of vascular distribution within subcutaneous tumors in nude mice (Figure 5A,5B) (23). In vivo, compared with the control group treated with exosomes derived from CAFs, the group injected with shMMP13 CAFs-derived exosomes resulted in a reduction in tumor volume (Figure 5C-5E). IHC analysis revealed that MMP13 expression was associated with decreased Ki67 and α-SMA expression (Figure 5F,5G). Exosomal MMP13 from CAFs was used in a xenograft model of subcutaneous tumorigenesis in C57BL/6 mice. Then, flow cytometry was used to examine the axillary lymph nodes of mice to investigate the effect of exosomal MMP13 on the TME (Figure S2A,S2B). The analysis of the types of immune cells in the axillary lymph nodes revealed that the shMMP13 CAFs-derived exosomes group had an increased percentage of CD4⁺ and CD8⁺ T cells, as well as a decreased percentage of B cells (Figure S2C). It is hypothesized that excessive degradation of ECM by MMP13 leads to an abnormal accumulation of fibrous matrix in lymph nodes, forming a dense fibrous network that hinders T cell infiltration into the tumor site, destroys B cell follicular structures, and affects B cell activation and antibody production. In parallel, activation of the Notch signaling pathway in the specified tissue was observed, which could be reversed upon inhibition of MMP13 expression (Figure 5H). These findings suggested that CAFs-derived exosomal MMP13 played a key role in NPC development as shown in Figure 5I. In conclusion, our investigation highlights the critical role of exosomal MMP13 in the activation of NFs to CAFs and NPC progression.

Figure 5.

MMP13 in CAFs-derived exosomes promote angiogenesis and progression of NPC in vivo. (A,B) Ultrasound Doppler shows the ratio of internal vascular distribution of subcutaneous tumors (circled area) in nude mice. (C) Tumorigenesis of CNE2 cells with CAFs shNC exosomes, shMMP13 CAFs exosomes. (D) The tumor size (means ± SEM) of different treatment groups. (E) Scatter graph of tumor volume and each dot represents the tumor volume of each mouse. (F) The ISH staining score was defined as low expression (scores of 0–8) or high expression (scores of 9–16) by the X-tile Software. (G) Representative IHC images of Ki67, α-SMA and CD34 were collected from different groups. The arrows indicate XXXXXXXXXXXX. (H) The expression levels of indicated proteins in tumor tissues from the mice were detected by Western blot. (I) The schematic illustrates that tumor exosome MMP13 promotes angiogenesis and proliferation in nasopharyngeal carcinoma by targeting Notch1 and Notch3 to promote the conversion of NFs to CAFs and reduce T cell production. *, P<0.05; **, P<0.01; ***, P<0.001. α-SMA, α-smooth muscle actin; CAF, cancer-associated fibroblast; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHC, immunohistochemistry; ISH, in situ hybridization; NF, normal fibroblast; NPC, nasopharyngeal carcinoma; SEM, standard error of the mean.

Discussion

In the present study, exosomes containing MMP13 were shown to transform NFs into CAFs. Importantly, the CAFs could in return improve NPC cell migration, proliferation, and tube formation. Our research revealed a novel potential molecular mechanism by which tumor cells and fibroblasts interact to accelerate the development of NPC.

Exosomes are membrane-enclosed vesicles that are produced by the majority of cells with a size range of ~40 to 160 nm (average ~100 nm) in diameter (4). Exosomes are thought to be crucial for intercellular communication and neoplastic transformation, according to convincing evidence (4). Numerous studies have highlighted the importance of exosomes released from NPC cells, which can interact with stromal cells through a variety of methods, facilitating NPC cells migration and proliferation as well as the angiogenesis process (24). In our earlier research, we discovered that NPC exosomes enhanced the metastatic capabilities of NPC cells (6). Duan et al. showed that exosomal miR-17-5p generated from NPC cells stimulates HUVEC angiogenesis via targeting BAMBI and controls AKT/VEGF-A signal (25). Exosomes packaged with LMP-1 activated CAFs contribute to tumor progression through autophagy and the coupling of stroma-tumor metabolism (26). Our earlier research revealed that human skin fibroblast (HSF) cells increased the amount of MMP13 they released into the environment, which helped NPC cells invade (6). According to the aforementioned data, NPC-derived exosomes stimulate growth and metastasis by controlling the microenvironment, specifically fibroblast cells. Nevertheless, it is still unclear how exosome-educated fibroblast cells aided in the development of NPC. In this study, we showed that exosomes from NPC cells could raise the levels of α-SMA and FAP expression in NF cells, suggesting that they might act as a trigger for the activation of NF into CAFs.

CAFs, which are the predominant stromal cells within tumors, can establish a particular microenvironment that promotes cancer growth, invasiveness, metastasis and therapy resistance (26). Numerous studies have shown the interactions between CAFs and cancer cells. However, CAFs could not be precisely categorized in our study and may need to be combined with single-cell sequencing (27). Cancer cells absorbed particular exosomes generated by CAFs, which aid in the development and metastasis by transferring a variety of substances (5). Exosomes derived from human and mouse lung cancer model cells were likewise discovered to have the ability to stimulate endothelial cells and fibroblasts, as well as to cause the stromal cells expressing angiogenic factors, thereby promoting angiogenesis in the lung cancer (28). Our research indicated that NFs with higher levels of α-SMA and FAP expression exhibited improved migratory ability after being cultured with exosomes from CNE2 as opposed to those produced by NP69. These results suggest that the activation of NFs into CAFs is mediated by exosomes from CNE2 cells.

However, the fundamental processes of CAF activation by cancer cells in NPC are yet unknown. Matrix metalloproteinases (MMPs) family belong to the zinc-dependent endopeptidase family (29). MMPs can accelerate the growth, invasion, and metastasis of malignant tumors by destroying various ECM constituents (29). MMP13, a significant MMP family member, is found in the cytoplasm (30). MMP13 is an oncogene seen in numerous malignant tumors, according to recent study (31). In previous study, we had demonstrated that exosomes purified from the peripheral plasma of NPC patients contain MMP13 (32). A statistical analysis of immunohistochemical MMP13 measurements revealed significant associations between high MMP13 expression and T-staging (P=0.043), lymph node metastasis (P=0.008), and clinical stage (P<0.01) in NPC patients (32). These findings imply that MMP13 expression in NPC samples could predict a poor prognosis prior to treatment. The precise function of MMP13 packed in exosomes in the tumor stroma has not been properly investigated, though. Here, we showed that MMP13 was transported from NPC cells to NFs via exosomes and stimulated NFs to CAFs for the first time. Our findings demonstrated that MMP13 was delivered to CAFs by exosomes produced from NPC cells, which enhanced CAFs’ capacity for migration. Notably, this study demonstrated that exosomal MMP13 produced from CAFs can promote angiogenesis, suppress T-cell immunity and support NPC development. Interestingly, several metalloproteinases are upregulated in Epstein-Barr virus (EBV) latent infection, indicating that the virus may be taking advantage of MMP13-mediated stromal remodeling, which is a distinct aspect of NPC pathogenesis (33). We hypothesized that integrins on the surface of exosomes could specifically bind to fibroblast membrane proteins and deliver MMP13 via receptor-mediated action. The incapacity of exosomes to activate CAFs when MMP13 is diminished corroborates the necessity for the delivery of MMP13, an extracellular protease that may be capable of directly cleaving the extracellular structural domains of Notch1 and Notch3 receptors in a process analogous to the mechanism by which MMP7 cleaves Notch1 (34), resulting in the generation of active XXXXX (ICD) structural domains capable of propagating to the nucleus and initiating target gene transcription. These need to be followed up on and validated. After being cultured with CAFs-secreted exosomes, the up-regulation of Notch1 and Notch 3 was especially relevant in NPC cells, we guessed that CAFs affect the biological characteristics of NPC cells through the Notch signal pathway.

Conclusions

In our previous study, we had found that MMP13, which is secreted by NPC cells, promotes the proliferation and migration of NPC. In the present study, overall, we first established that MMP13 can activate NFs into CAFs in exosomes released by NPC cells. Compared to NFs, CAFs are better able to proliferate and migrate. CAFs secrete large amounts of pro-inflammatory cytokines that act directly on tumor cells, inducing cell proliferation, resistance to cell death, and epithelial-mesenchymal transition (EMT). Our research concentrated on the function of CAFs and MMP13. Exosomes derived from CNE2 cells with MMP13 knocked down can lessen the ability of CAFs to migrate and proliferate. We found that MMP13 in CAFs promoted angiogenesis and remodeled the TME, resulting in an elevated ratio of CD4⁺ T cells and CD8⁺ T cells. Our research then showed that MMP13 may play a significant role in the activation of CAFs by activating the Notch signal pathway. We defined the underlying mechanism of crosstalk between NPC cells and NFs to stimulate tumor growth via exosomal MMP13, which may support effective NPC prevention and therapy approaches.

Supplementary

The article’s supplementary files as

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was performed in line with the principles of the Declaration of Helsinki and its subsequent amendments. Approval was granted by the Ethics Committee of Affiliated Hospital of Nantong University (IRB No. 2017-L078), and each patient was fully informed and signed a consent form before performing the experiments. Animal experiments were performed under a project license (No. 20190304-005) granted by the Institutional Animal Care and Research Advisory Committee of Nantong University and in compliance with institutional guidelines for the care and use of animals.

Data Sharing Statement

Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-428/dss

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DOI: 10.21037/tcr-2025-428